Synthesis, biofilm formation inhibitory, and inflammation inhibitory activities of new coumarin derivatives

Coumarins are heterocycles of great interest in the development of valuable active structures in chemistry and biological domains. The ability of coumarins to inhibit biofilm formation of Gram positive bacterium (Staphylococcus aureus), Gram negative bacterium (Escherichia coli) as well as the methicillin-resistant S. aureus (MRSA) has been previously described. In the present work, new hybrid coumarin-heterocycles have been synthesized via the reaction of coumarin-6-sulfonyl chloride and 6-aminocoumarin with different small heterocycle moieties. The biological efficacy of the new compounds was evaluated towards their ability to inhibit biofilm formation and their anti-inflammatory properties. The antimicrobial activities of the newly synthesized compounds were tested against Gram positive bacterium (S. aureus ATCC 6538), Gram negative bacterium (E. coli ATCC 25922), yeast (Candida albicans ATCC 10231) and the fungus (Aspergillus niger NRRL-A326). Compounds 4d, 4e, 4f, 6a and 9 showed significant MIC and MBC values against S. aureus, E. coli, C. albicans, and methicillin-resistant S. aureus (MRSA) with especial incidence on compound 9 which surpasses all the other compounds giving MIC and MBC values of (4.88 and 9.76 µg/mL for S. aureus), (78.13 and 312.5 µg/mL for E. coli), (9.77 and 78.13 µg/mL for C. albicans), and (39.06 and 76.7 µg/mL for MRSA), respectively. With reference to the antibiofilm activity, compound 9 exhibited potent antibiofilm activity with IC50 of 60, 133.32, and 19.67 µg/mL against S. aureus, E. coli, and MRSA, (respectively) considering the reference drug (neomycin). Out of all studied compounds, the anti-inflammatory results indicated that compound 4d effectively inhibited nitric oxide production in lipopolysaccharide-(LPS-) stimulated RAW264.7 macrophage cells, giving NO% inhibition of 70% compared to Sulindac (55.2%)

The 1 H NMR spectra were utilized to confirm the formation of the newly coumarin derivatives 5a,b.For example, the 1 H NMR (DMSO-d 6 ) spectrum of 5a displayed two singlet signals at δ 10.72, 10.22 ppm supporting the presence of two protons of NH, besides a doublet signal at δ 6.59 (J = 8.3 Hz) authorized the presence of H-2 of coumarin (Fig. s12).

Antimicrobial activity
Using the agar well diffusion assay, the newly synthesized coumarin derivatives, were estimated for their antimicrobial activity towards S. aureus (ATCC 6538), E. coli (ATCC 25933), C. albicans (ATCC 1023) besides A. niger (NRRL-A326) 35 .It has been found that the newly synthesized coumarin derivatives exhibited diverse activities in relation to the test microbe (Table 1, Fig. s1).Compounds 4f and 9 had considerable antimicrobial activities against all test microbes with inhibition values of 16 and 16 mm against S. aureus, 15 and 9 mm against E. coli, 18 and 17 mm against C. albicans, and 15 and 19 mm against A. niger.It has been also found that compounds 4d, 4e, 6a, and 7 had moderate activities against S. aureus with inhibition values of 13, 10, 14, and 12 mm (respectively), whereas the other compounds exhibited low or no activities against the same test microbe.For E. coli, compounds 4b, 5b and 6b showed low activities with inhibition values of 7, 8 and 7 mm (respectively) and the other compounds showed negative results.On the other hand, compound 4d had high activity with C. albicans

Inhibition of biofilm formation
Microorganisms that can produce biofilms are known to be one of the major factors contributing to antibiotic resistance.Therefore, many experiments have been conducted to overcome these serious problems by searching for new drugs that can prevent biofilm formation 38 .S. aureus is one of the most frequent causes   24,39,40 .
Coumarin and its derivatives have attracted the attention of many microbiologists due to their antimicrobial effectiveness 41,42 .There are also further studies on the effectiveness of coumarin as an inhibitor of biofilm formation 20,43 .A recent study has proven that 3-hyrdroxy-coumarin, a marine bacterium-derived compound, showed antibiofilm formation 44 .So, the inhibition of biofilm formation was performed for the five most active compounds 4d, 4e, 4f, 6a, and 9 using neomycin as a reference control compound.Table 3 and Fig. s3 explained the ability of the most active compounds with potent antibiofilm formation expressed as IC 50 values.It was found that, compound 9 exhibited the best antibiofilm activities against S. aureus, E. coli, and MRSA with IC 50 values of 60, 133.32, and 19.67 µg/mL, respectively in comparison to neomycin (IC 50  = 19.67,79.289, and 39.34 µg/ mL, respectively).For the other compounds, it has been reported that compounds 4d and 6a had considerably acceptable results with S. aureus (IC 50 of 185.51 and 355.52 µg/mL, respectively).The other compounds had appreciable IC 50 values against the same test microbe.For E. coli, compounds 4d and 4e showed noticeable IC 50 values 321.25 and 345.40 µg/mL (respectively).All the other compounds (4d, 4e, 4f and 6a) when tested as antibiofilm formation by MRSA, compound 4e and 6a showed promising results (IC 50 : 85.02 and 40.73 µg/mL, respectively) in respect to neomycin.Out of the five selected compounds for inhibition of biofilm formation, compound 9 was the potent one and showing antibiofilm activity against MRSA (about twofold more potent) and about fivefold and threefold lower against S. aureus, E. coli, respectively.

Effect of compounds on nitric oxide levels in LPS-stimulated RAW 264.7 macrophages
Coumarins represent an important family of oxygen-containing heterocycles, widely distributed in nature 45,46 .Coumarin and its derivatives exhibited a broad range of biological and pharmacological activities 47 .A previous study indicated that imperatorin (a coumarin derivative) has an anti-inflammatory effect in lipopolysaccharidestimulated mouse macrophages (RAW264.7) in an in-vitro model of edema, as it inhibits the protein expression of nitric oxide synthase (NOS) and a cyclooxygenase-2 (COX-2) 48 .The effect of the active compounds (4d, 4f, 6a, and 9) on levels of Nitric Oxide (Fig. 2A) in LPS-stimulated RAW 264.7 cells was investigated according to the method of Elshahid et al. 49 .All the cells were treated with the studied compounds along with LPS or LPS alone for 24 h.To determine the level of NO production, the released of nitrite into the culture medium was measured using Griess reagent.As a result, LPS alone markedly induced NO production compared with that generated by  the control.However, pretreatment with the studied compounds affected NO levels that significantly produced in LPS-stimulated RAW 264.7 cells as shown in Fig. 2A.Moreover, compounds (4d) induce marked inhibition on NO production by (70%) as compared to LPS (Table 4).
In a parallel experiment, to examine the cytotoxicity of the studied compounds on RAW 264.7 cells, the cells were treated with each compound for 24 h in the presence or absence of LPS, and the cytotoxic potential was measured by the MTT assay 50 .
The results showed that compound (4d) was the least cytotoxic compound (≈ 20% cytotoxicity) indicating high cell viability.Meanwhile, compound (4f) and Sulindac (positive control) showed higher cytotoxic effect as indicated by the MTT reduction assay (Fig. 2B).These results clearly indicate that the anti-inflammatory activity of 4d in LPS-stimulated RAW 264.7 macrophages was not due to direct cell death.Accumulating evidence indicates that NO is a critical mediators of inflammation 51,52 .NO plays a pivotal role in many body functions; however, its overproduction, particularly in macrophages, can lead to cytotoxicity, inflammation, and autoimmune disorders 51,52 .Our data are in agreement with several in-vitro studies performed with LPS-stimulated RAW264.7 cells, which showed that coumarin and its derivatives have shown a therapeutic effect against edema, eliminating proteins and fluid from injured tissue by activating mechanisms such as phagocytosis, enzyme release, and proteolysis 48,53,54 .

Experimental part
Chemistry All reagents and solvents were of commercial grade.Coumarin (Sigma-Aldrich Chemie GmeH, Taufkirchen, Germany).Melting points of the synthesized coumarins were measured on the digital melting point apparatus (Electro thermal 9100, Electro thermal Engineering Ltd., serial No. 8694, Rochford, United Kingdom) and are uncorrected.A Bruker Avance spectrometer (Bruker, Germany) was used to measure the 1 H and 13 C NMR spectra of new coumarins at 500 and 125 MHz, respectively.Elemental analyses were carried out on a Perkin-Elmer 2400 analyzer (USA) and were found within ± 0.4% of the theoretical values.TMS was used as the internal standard and hydrogen coupling patterns are described as (s) singlet, (d) doublet, (t) triplet, (q) quartet and (m) multiple.Chemical shifts were defined as parts per million (ppm) relative to the solvent peak.
General Procedure for the preparation of coumarin sulfonamide derivatives 4a-f.An equal proportion of coumarin-6-sulfonyl chloride and applicable amino-compounds (10 mmol) in absolute ethanol (10 mL) was refluxed under stirring for 5-30 min.The precipitate formed on hot was collected by filtration and recrystallized from the proper solvent.

Antimicrobial assay
The antimicrobial activity of the synthesized compounds were assessed against Staphylococcus aureus ATCC 6538-P as Gram positive bacterium, Escherichia coli ATCC 25933 as Gram negative bacterium, Candida albicans ATCC 10231 as yeast as well as the filamentous fungal test microbe Aspergillus niger NRRL-A326 by the agar well diffusion method 35 .Bacterial and yeast test microbes were inoculated on nutrient agar medium plates seeded with 0.1 mL of 10 5 -10 6 cells/mL whereas the fungal test strain was cultivated on plates having potato dextrose agar medium that seeded by 0.1 mL (10 6 cells/mL) of the fungal inoculum.5 mg of each sample was dissolved in 2 mL of DMSO. 100 µl from each sample were distributed in holes developed in each inoculated plate.Then plates were kept at 4 °C for more than 2 h to allow extreme dispersion.The plates were then kept at 37 °C overnight for bacteria and yeast and kept at 30 °C for 2 days for the fungus in vertical location to permit maximum microbial growth.Neomycin was used as reference drug for Gram-positive and Gram-negative bacteria as well as yeast.Cyclohexamide was used as reference drug for fungi (A.niger).The clear zone diameters expressed in millimeter (mm) were used to differentiate the antimicrobial activity of tested compounds.The experiment was carried out twice and their mean were considered.

Evaluation of minimum inhibitory concentration (MIC) and Minimum bactericidal Methicillin Resistant S. aureus concentration (MBC)
MIC was performed using S. aureus ATCC 6538, Gram-positive bacterium, and E. coli ATCC 25922, Gramnegative bacterium, Candida albicans ATCC 10231 as yeast, and Methicillin Resistant S. aureus (MRSA) as tested microbes that are grown on a Mueller Hinton medium.Test microbes were cultivated in 100 mL bottles with each test at 35 °C for 24 h.Cells were obtained by centrifugation (4000 rpm) under a sterile condition at 4 °C for 15 min.The cells were washed using sterile saline until the supernatant was clear.Cells with an optical density of 0.5 to 1 (at 550 nm) giving an actual number of colony-forming units of 5 × 10 6 cfu/mL were obtained.
Resazurin solution was prepared by dissolving 270 mg tablet in 40 mL of sterile distilled water.Then, 96-well sterile microplates were prepared.Then, 50μL of test material in DMSO was pipetted into the first row of the plate.To all other wells, 50μL of broth medium was added.Two-fold serial dilutions were performed.Then, 10μL of resazurin indicator solution was added, 10μL of bacterial suspension was added to each well.The plates were prepared in duplicate and placed in an incubator set at 37 °C for 18-24 h.Any colour changes from purple to pink or colourless were recorded as positive.The lowest concentration at which colour change occurred was taken as the MIC value.MBC has been performed by streaking of the two concentrations higher than MIC and the plates exhibiting no growth were considered as MBC 35,36 .Neomycine has been used as positive control 55 .

Inhibition of biofilm formation (crystal violet method)
Bacterial strains were incubated in test tubes with TSB (5 mL) containing 2% w/v glucose at 37 °C for 24 h.After that, the bacterial suspensions were diluted to achieve turbidity equivalent to a 0.5 McFarland standard.The diluted suspension (2.5μL) was added to each well of a single cell culture polystyrene sterile, flat-bottom 96-well plate filled with TSB (200μL) with 2% w/v glucose.Sub-MIC concentration values of compounds 4d, 4e, 4f, 6a, and 9 were directly added to the wells to reach concentrations ranging from 100 to 0.1 μM to assess BIC 50 values that are, the concentration at which the percentage of inhibition of biofilm formation is equal to 50%.
Plates were incubated at 37 °C for 24 h.After biofilm growth, the content of each well was removed, wells were washed twice with sterile NaCl 0.9% and stained with 200μL of 0.1% w/v crystal violet solution for 15 min at 37 °C.The excess solution was removed, and the plate was washed twice, using tap water.A volume of 200 μL of ethanol was added to each stained well to solubilize the dye 35,38 .Neomycine has been used as positive control 55 .Optical density (O.D.) was read at 600 nm using a microplate reader (GloMax®-Multi Detection System, Milan, Italy).The experiments were run at least in triplicates, and three independent experiments were performed.The percentage of inhibition was calculated through the formula:

Anti-inflammatory assay
Cell culture (seeding and treatment) The RAW 264.7 macrophage cell line were supplied from ATCC (American type culture collection).The cells were sub-cultured in Roswell Park Memorial Institute's RPMI 1640 medium 49 .

Procedure
The following procedures were all completed in a biosafety level II Laminar flow cabinet in a clean environment.RAW 264.7 cells were suspended at concentration of 1 × 10 5 cells per well (in 96 well plates).The cells were then incubated with the test compounds, LPS (lipopolysaccharide, negative control) and Sulindac (positive control drug) according to the method 49 .After 24 h, the supernatant was gently transferred to new 96-well plates for measuring nitric oxide (NO) while cells were used for cell viability testing using the MTT method.The percentage change in viability was calculated according to the below formula

Nitric oxide assay
The generation of nitric oxide (NO) was measured in the supernatants of cultivated RAW 264.7 cells.With slight modification, the Nitric Oxide (NO) measurement was carried out as described by Eid et al. 50using the Griess reagent.In detail, 50 μl of cell culture media were added to 50 μl of Griess reagent then incubated at room temperature for 15 min before being measured at 540 nm 50 .A sodium nitrite standard curve was used to calculate the amount of nitrite, as shown in equation:

Figure 2 .
Figure 2. (A) Effects of the studied compounds on the production of nitric oxide (NO) in LPS-stimulated RAW264.7 macrophages.Cells were treated with the studied compounds at concentration 100 µg/mL plus LPS (1 μg/mL) or LPS alone for 24 h.Sulindac (NSAID) was used as a positive control.(B) Cytotoxic effect of the studied compounds and Sulindac on Raw-264.7 macrophages at concentrations (100 μg/mL).Values are expressed as the means ± SD (n = 3) p < 0.0001 (versus LPS alone, 2A).

Table 1 .
In vitro antimicrobial activity of the newly synthesized coumarin derivatives against different test microbes using agar well diffusion method at concentration (250 μg/100μL).

Table 3 .
Inhibition of biofilm formation (IC 50 µM) from S. aureus, E. coli and MRSA cultures treated with